Composition of biocompatible microparticles of alginic acid for the controlled release of active ingredients by intravenous administration

ABSTRACT

Composition of biocompatible microparticles of alginic acid for the controlled release of active ingredients by intravenous administration. The invention relates to a biocompatible composition which comprises microparticles of alginic acid or its salts and an active ingredient. More particularly, the invention relates to microparticles for the encapsulation of an active ingredient to be administered intravenously to a patient who needs it. These microparticles are of a combination of size sufficient to increase the half-life or survival of the active ingredient in blood, with a low uptake in the liver and a fast cell clearance when administered intravenously.

The present invention relates to a biocompatible composition whichcomprises microparticles of alginic acid or its salts and an activeingredient. More particularly, the present invention relates tomicroparticles for the encapsulation of an active ingredient to beadministered intravenously to a patient who needs it. Thesemicroparticles show a combination of size adequate to increase thehalf-life or survival of the active ingredient in blood, with a lowuptake in the liver and a fast cell clearance when administeredintravenously. The active ingredient in the composition of the presentinvention can be a peptide, protein or hormone, of human or animalorigin, of natural, recombinant or transgenic nature. Included inexamples of active ingredients in the composition of the presentinvention are blood clotting factors, such as factor VIII, factor IX orfactor VIIa.

BACKGROUND OF THE INVENTION

The increase in the half-life in the blood of a therapeutic activeingredient has advantages, including fewer administrations beingnecessary to gain the desired therapeutic effect. This reduction in thenumber of administrations is of special importance in drugs forparenteral administration, most especially in those for intravenous useand of special relevance to long-term medications such as, for example,those for the treatment of chronic disorders.

The current tendency is, as far as possible, to administer activeingredients by routes which do not need intravenous access, because ofcomplexity and inconvenience for the patient when this method is used.However, there is a series of active ingredients for which there is atpresent no alternative to intravenous administration. Included in theseare active ingredients of great size and complexity, such as biologicalor biotechnological products, which include proteins and hormones.

One example of a chronic therapeutic condition where the repeatedintravenous administration of complex active ingredients is necessary ishaemophilia. Haemophilia is a hereditary disease featuring theappearance of internal and external bleeding due to the total or partialdeficiency of proteins related to the clotting of blood. Haemophilia Afeatures a deficiency of clotting Factor VIII, which impedes the normalgeneration of thrombin, making it difficult for the blood to clotnormally as a result. In the case of haemophilia B, the deficiency ofFactor IX causes a similar clinical state.

For the treatment of haemophilia, the first therapeutic option consistsof replacing the absent protein (FVIII or FIX) by the administration ofa therapeutic concentrate containing this factor. Another therapeuticoption to obtain correct haemostasis in haemophilia is theadministration of FVIIa, which has the ability to generate thrombin inthe absence of FVIII or FIX. However, this type of treatment is usuallylimited to cases where treatment with FVIII or FIX is problematic or hasproved ineffective, such as for example in patients who have had aninhibitory immunological response to these active ingredients. To date,none of these products has been successfully administered by any methodof administration except intravenous, given its structural complexityand low epithelial permeability.

Therefore, patients affected by haemophilia require intravenousadministrations repeated with a frequency determined by its half-life inthe plasma. In the case of FVIII the half-life is about twelve hours.This implies, according to the monograph of the World Federation ofHaemophilia (Casper, C K, Hereditary Plasma Clotting Factor Disordersand Their Management 5th ed. WFH, Sam Schulman Ed., 2008), that for aprimary prophylaxis regime, i.e., for the prevention of bleeding inchildren without articular damage a dose of about 20 U/kg every 48 hoursis used, sufficient to maintain a level of plasma FVIII of more than 1%of the normal value. Essentially, this treatment changes a person withsevere haemophilia into one with slight or moderate haemophilia. In thecase of FIX, the half life is about 26 hours, so that for primaryprophylaxis doses of about 40 U/kg twice a week can be administered inorder to maintain a minimum level of 1%.

It has to be taken into account that prophylaxis from an early age(about age one year or at the start of crawling) is the standard of carerequired in order to avoid articular damage in cases of severehaemophilia.

Consequently, haemophilia is a clear example where an increase in thehalf-life of the active ingredient would provide a substantialimprovement in the patient's quality of life, as it would reduce thenumber of intravenous administrations, especially difficult in childrenof a young age.

Other examples of long-term treatments with intravenous administrationproducts are for example, the use of immunoglobulins (IgG) in primaryimmunodeficiencies and the use of antithrombin III (AT) and alpha-1antitrypsin (AAT) in congenital deficiencies.

There are numerous technological approaches aiming at extending theplasma half-life of these types of active ingredients. One of the moststudied has been the derivatisation of proteins with compatiblepolymers, as is the case of polyethylene glycol (PEG). This technologyconsists of the practice of carrying out a chemical reaction to join PEGchains covalently to protein amino acids. This technique has proveduseful in the case of hormones and peptide chains of small size, such asinterferon, since for compounds of this type the principal mechanism ofelimination is renal clearance, easily controllable by a simple increasein size (Bailon Pascal et al, Bioconjugate Chem. 2001, 12, 195-202).However, it is still to be decided whether it can be used in morecomplex active ingredients, as they are based on the externalmodification of the protein structure to be treated. In addition,covalent bonds of this type with the protein considerably reduce thebiological activity of the treated hormone or protein.

Another alternative to modify the half-life has been the addition ormodification of the sugar residues naturally present in proteins orhormones (Perlman Signe et al, The Journal of Clinical Endocrinology &Metabolism 88 (7): 3227-3235, 2003). This procedure claims to alter theprotein, by modifying its recognition by the receptors involved in itsdegradation. However, the inherent risks of this alteration are obvious,given the high immunogenic potential of the glycosylations present inthe proteins.

A third line of action has been to obtain chimeric proteins where theactive sequence of a protein of interest is expressed, bonded tosequences of plasma proteins which have a considerable half-life, as isthe case of albumin or fragments of immunoglobulins (Dennis, Marks S. etal, The Journal of Biological Chemistry vol. 277, No. 38, Issue ofSeptember 20, pp. 35035-35043, 2002). However, this technology has asits principal disadvantage, in addition to the expected immunogenicityassociated with exposing patients to proteins not present in nature,loss of efficacy of the protein upon the modification of its structurein such a dramatic way.

Another possibility investigated to extend the half-life of complexactive ingredients has been the co-administration of the product with aliposome stabilised with PEG. This technique is based on the affinity ofthe active ingredient for PEG, which allows a reversible associationbetween the protein and the liposome. This transitory association mustprovide an increase in the half-life of the active protein ingredient,since liposomes stabilised with PEG stay in circulation for a long time.However, it has not been possible to corroborate this hypothesis inpractice, as this system has proved to be ineffective in extending thehalf-life of FVIII in haemophilia patients (Powell J. S et al, Journalof Thrombosis and Haemostasis, 6: 277-283, 2007).

To date, no system amongst those previously described has been able tosignificantly modify the half-life, with the exceptions described wherethe introduction of structural modifications and alterations make theirapplication unviable or very complex for the treatment of humanpathologies.

The controlled release of therapeutic agents encapsulated inbiodegradable polymeric microspheres has been extensively studied. Themicroencapsulation of the active ingredient in biodegradable polymersallows the release of the drug to be controlled. This approach hasrecently been applied in controlled release formulations forsubcutaneous use based on derivatives of lactic and glycolic acids.These formulations have been used successfully in the encapsulation of awide variety of active ingredients, including cytostatics,anti-inflammatories, peptides and hormones, inter alia (Tamilvanan S. etal, PDA Journal of Pharmaceutical Science and Technology, vol. 62, No.2, March-April 2008 pp. 125-154).

Pankaj (U.S. Pat. No. 5,417,982) describes the use of lactic andglycolic acid microspheres for the controlled release of hormones byoral administration. Although Pankaj describes the possibility ofobtaining an injectable product, it is very unlikely that this inventioncan be administered intravenously, given the requirements of this methodof administration, and in any case, this invention does not anticipatethe use of alginates for this purpose.

Sivadas (Sivadas Neeraj et al, International Journal of Pharmaceutics358 (2008) pp. 159-167) describes the use of different polymers,including hydroxypropyl cellulose, chitosan, hyaluronic acid, gelatine,ovalbumin and glycolic polylactic acid, as vehicles for theencapsulation of proteins for their administration by inhalation.

One disadvantage of the use of lactic and glycolic acid derivatives isthe need to make the preparations in the presence of organic solvents,some of them of known toxicity, such as polyvinyl alcohol, which exhibitincompatibilities with the conservation of the biological activity ofcomplex active ingredients such as proteins and hormones.

The use of these polymers also results in highly hydrophobic particles,which, as is discussed below, are rapidly eliminated from thecirculation by cellular uptake mechanisms. An additional disadvantage isthe creation of a locally very acid environment around the particle atthe time of its dissolution and, therefore, at the time when the activeingredient is released. This is due to the fact that the polymerdecomposes in lactic acid and glycolic acid, which creates an extremelyacidic environment around the particle in dissolution. It is this acidenvironment which can damage sensitive active ingredients andparticularly those which have complex amino acid structures with labilebiological activity.

Alginates have many applications in the food and pharmaceuticalindustries and in the chemical industry in general. This wide variety ofapplications is defined by their hydrocolloid property, i.e., theirability to hydrate themselves in water so as to form viscous solutions,dispersions or gels. This feature gives alginates unique properties asthickening agents, stabilising agents, gelling agents and film formers.

One area where the properties of alginates have been widely exploitedhas been in the encapsulation of active ingredients in particular inorder to improve their solubility, or to assist the administration ofdrugs (Tønnesen, Hanne Hjorth et al, Drug Development and IndustrialPharmacy, 28(6), 621-630 (2002)) by various routes. Amongst these is theuse of oral administration given the mucoadhesive properties ofalginate. The subcutaneous method has also been examined. However thereis no history of intravenous use due to the strict requirements of thisroute of administration.

For example, Benchabane (Benchabane, Samir et al, Journal ofMicroencapsulation, September 2007; 24(6): pp. 565-576) describes theuse of alginates in the production of albumin microcapsules by“spray-drying” for oral administration. In a similar antecedent, Coppi(Coppi, Gilberto et al, 2001, Drug Development and Industrial Pharmacy,27(5), pp. 393-400) demonstrates the formation of microspherescrosslinked with calcium and chitosan for the oral administration ofproteins. In both cases, alginate acts as a protector of protein againstthe proteolytic degradation which occurs naturally during gastricdigestion.

Further, Mladenovska (Mladenovska, K., International Journal ofPharmaceutics 342 (2007) pp. 124-136) describes obtaining microparticlesof alginate/chitosan for colonic administration.

Sivadas (Sivadas Neeraj et al, International Journal of Pharmaceutics358 (2008) pp. 159-167) also mentions the use of alginates as a vehiclefor the encapsulation of proteins for administration by inhalation.

Apart from the direct administration of active ingredients, alginateshave also been suggested as vehicles for the administration of complextherapeutic forms. For example, in patent WO 2006/028996 A2 the use ofalginate and Emulsan for the transport of detoxifying agents ofbacterial toxins is described.

Another example is the use of alginate in the encapsulation ofmultivesicular liposomes (Dai, Chuanyun, et al, Colloids and Surfaces B:Biointerfaces 47 (2006) pp. 205-210) or live cells (European Patent,publication number: 2 159 523). In this case, the administration of livecells has as its objective their application in regenerative medicine orgene therapy (WO 2007/046719 A2; Peirone, Michael et al, J. Biomed.Mater. Res. 42, pp. 587-596, 1998; García-Martín, Carmen et al, TheJournal of Gene Medicine, J Gene Med 2002; 4: pp. 215-223). Curiously,García-Martín (García-Martín, Carmen et al, The Journal of GeneMedicine, J Gene Med 2002; 4: pp. 215-223) describes the possibleapplication of the administration of genetically modified live cells forthe treatment of haemophilia A, exemplifying the medical relevance ofthe problem. In this case, alginate microcapsules which contain livecells are implanted intraperitoneally by the introduction of a catheter.In this case, both the objective of the treatment and the method ofadministration—non-intravenous—are radically far from the presentinvention.

In spite of this wide experience in the use of polymers for theencapsulation of complex active ingredients, such as proteins, there areno references which can resolve the problems associated with theintravenous administration of these products. As Wong et al describe(Wong, Joseph et al, Advanced Drug Delivery Reviews 60 (2008) pp.939-954) there are only three approved products which use particlesuspensions for their intravenous administration. None of them includethe use of alginates in their composition. In all cases, an increase inhalf-life is not sought, but an improvement in the solubility of theproduct.

The difficulty of effectively administering microparticles intravenouslycan be expressed in (a) the basic aspects of design of the product, suchas the size of the particle and distribution, absence of organicsolvents, and also the homogeneity, viscosity and “syringeability” ofthe suspension—understanding as “syringeability” the ease of suction andinjection of the product; (b) the technical aspects of production andpreparation on an industrial scale, such as the uniformity of the dose,the unwanted crystallisation of salts in the case of products obtainedby solvent precipitation, the sterility and apyrogenicity of theproduct; and (c) biological aspects, such as the non-deliberatealteration of the pharmacokinetic and pharmacodynamic profile,alteration of the biodistribution, the bioaccumulation of the polymer,phagocytic activation, toxicity and effects of embolisation oractivation of the complement.

In this connection, one of the most significant problems in thedevelopment of these products is its fast clearance by the mononuclearphagocyte system (MPS), previously called reticuloendothelial system(RES), which includes all the cells derived from the monocyticprecursors of the bone marrow, the monocytes of the peripheral blood andthe macrophages or histiocytes of the various organs and tissues.Amongst the latter must be mentioned, because of their importance in theclearance of microparticles in plasma, the Küpfer cells of the liver andthe macrophages distributed in the spleen and the bone marrow(Passirane, Catherine et al, Pharmaceutical Research, Vol. 15, No. 7,1998 pp. 1046-1050).

It has been widely described that after the intravenous administrationof nano- or micro-particles they are rapidly opsonised by the proteinsof the plasma. These proteins absorbed in their surface inducerecognition and uptake by the MPS cells (Passirane, Catherine et al,Pharmaceutical Research, Vol. 15, No. 7, 1998 pp. 1046-1050).

A similar effect has been observed in liposomes (Ishida, Tatsuhiro etal, Journal of Controlled Release 126 (2008) pp. 162-165), where aphenomenon known as Accelerated Blood Clearance (ABC) has beendescribed. Both in the case of polymeric microparticles and in that ofliposomes, the opsonisation phenomena are also directly related to theactivation of the complementary system (Ishida, Tatsuhiro et al, Journalof Controlled Release 126 (2008) pp. 162-165; Koide, Hiroyuki et al,International Journal of Pharmaceutics 362 (2008) pp. 197-200).

In practice, this phagocytosis phenomenon prevents the development ofdrugs with an extended half-life based on microparticles administeredintravenously, since the increase in size associated with encapsulationdoes not just increase but on occasions causes accelerated degradation.Obviously, this phenomenon is only observed by means of in vivoexperimentation, which involves studies of pharmacokinetics in animals.

The relationship between this clearance via phagocytosis and the size ofthe particle has been widely documented. Champion (Champion, J A, PharmRes. 2008 August; 25(8): 1815-21. Epub 2008 Mar. 29) specificallydescribes the relationship between the phagocytosis experienced bypolymeric microparticles and their size, observing a maximum effectbetween 2-3 μm. Other features which define the uptake of microparticlesby the MPS in vivo are the hydrophobicity of the particles and theirZeta Potential (Z Potential) (Szycher, Michael, High PerformanceBiomaterials: A Comprehensive Guide to Medical and PharmaceuticalApplications, published by CRC Press, 1991 ISB 0877627754,9780877627753, 812 pages).

Z Potential is a property of the particles. Specifically, disperseparticles tend to become electrically charged by the adsorption of ionsfrom the external phase, or by ionisation of functional groups on theirown surface. One consequence of this is that a layer of counterionscalled the Stern layer will appear back to back with the particle in theenvironment of a negatively charged dispersed particle. A diffused layerappears on said stern layer featuring the presence of mobile charges (ofboth signs) which will counteract the charge of the particle, as afunction of the distance to the same. Z Potential is what we call thedifference in potential between the layer of counterions and the pointof electrokinetic neutrality.

Z Potential values are crucial for the stability of the majority ofdispersed systems, since the latter will regulate the degree ofrepulsion between dispersed particles of similar charge, preventing saidparticles from coming too close to one another and the forces ofinter-particle attraction, caused by the coalescence phenomena, frombecoming predominant. As regards the Z potential, it has been disclosed(Szycher, Michael, High Performance Biomaterials: A Comprehensive Guideto Medical and Pharmaceutical Applications, published by CRC Press, 1991ISB 0877627754, 9780877627753, 812 pages) that partially negative Zpotentials close to 0 reduce phagocytosis.

Moreover, hydrophobicity also assists the opsonisation and uptake of theparticles. This is of particular interest, since particles derived frompolylactic and glycolic acids are, for example, highly hydrophobic.

One approach achieved to extend the half-life in plasma ofmicroparticles and liposomes was the introduction, onto the surfacethereof, of charged polymers which are able to modify their charge andgenerate a hydrophilic surface layer to protect them from opsonisationand phagocytosis. Amongst them is the use of polyethylene glycol (PEG)(Ishida, Tatsuhiro et al, Journal of Controlled Release 126 (2008)162-165; Owens III, Donald E et al, International Journal ofPharmaceutics, volume 307, Issue 1, 3 Jan. 2006, Pages 93-102) orheparin (Passirane, Catherine et al, Pharmaceutical Research, Vol. 15,No. 7, 1998 pp. 1046-1050).

This approach complicates and makes difficult the development of apharmaceutical product because of the increase in the complexity of thesystem. In addition, as has been previously discussed, the use ofPEG-liposomes has proved to be ineffective in extending the half-life ofa complex protein such as FVIII (Powell J. S et al 2007, Journal ofThrombosis and Haemostasis, 6: pp. 277-283).

In the case of microparticles, in order to obtain a viable product forintravenous administration it would be necessary to have hydrophilicparticles with a suitable combination of size and Z potential.

Terrence (European Patent, Publication Number: 2 286 040, EuropeanApplication Number: 00973477.3) describes the use of polymers as asystem of administration capable of increasing the half-life of theactive encapsulated ingredients. For this purpose, this inventionrequires the use of (1) a first water-soluble polymer, (2) at least oneanionic polysaccharide as first complexing agent and (3) a divalentcation as a second complexing agent. As has been observed, the inventionmentioned is technically complex and difficult to use in practice. Incontrast, in the present invention the controlled release of the activeingredient is achieved with far simpler microparticles, which involvethe use of a single polymer that possesses all the properties necessaryfor its application. Furthermore, Terrence's invention does notdemonstrate the compatibility of its preparation for intravenous use bysize, or explain or illustrate how to avoid cellular phagocytosis.

Alginate, unlike other polymers with PLA or PLGA, is hydrophilic.Particles generated in the present invention have been shown to havepartially negative Z potentials sufficient to prevent the aggregation ofparticles, but neutral enough to provide a low opsonisation profile.

The maximum sizes of particle acceptable for intravenous administrationare around 5 μm. This is demonstrated by the existence of registereddrugs which use albumin marked for diagnosis by ultrasounds (Optison,data sheet 28) with an average size of 3.0-4.5 μm.

Alginate is biocompatible, and has been used extensively for oraladministration in humans, given its wide use in the food industry. Wheninjected intravenously as a non-particulate polymer, it is eliminated ina biphasic form with half-lives of 4 and 22 hours (Hagen, A. et al,European Journal of Pharmaceutical Sciences, Volume 4, Supplement 1,September 1996, pp. 100-100 (1)) without adverse effects being observed.Alginate is eliminated via urine.

In addition, the fact that it is a water-soluble polymer assists itscompatibility with complex proteins, as these latter are its naturalsolvent.

The present invention relates to a composition comprising microparticlesof alginic acid or its pharmaceutically acceptable salts by which acontrolled release is achieved, and achieves an increase in thehalf-life of the active ingredients administered intravenously, andresults in a lower frequency of application and achieves more stablelevels of active ingredient in the blood, thus potentially reducing thepeaks and troughs typical in the concentration of the active ingredient,which occur as a result of the periodical infusion of the same.

The present invention describes hydrophilic microparticles of alginatewith a combination of size suitable for intravenous infusion andphysio-chemical characteristics suitable for preventing the rapidphagocytosis of the same, allowing a controlled release of complexactive ingredients.

DESCRIPTION OF THE INVENTION

Alginic acid and its salts (ammonium alginate, calcium alginate,potassium alginate, sodium alginate and propylene glycol alginate) areamong the polymers most used and studied in the encapsulation of activeingredients due to their physicochemical and biochemical properties.They are polysaccharides of natural origin, commercially produced fromalgae or bacteria.

Alginates are alginic acid salts, a linear polysaccharide made up of twomonomer units, β-(1-4)-D-mannuronic (M) acid and α-(1-4)-L-guluronic (G)acid. These are grouped in blocks forming a wide variety of sequences,the most common being G, M and MG.

In the presence of multivalent cations like calcium (Ca⁺⁺), strong bondsare made between contiguous G blocks forming an extended network ofalginates. Calcium ions are situated as bridges between the groups witha negative charge of guluronic acid. In some formulations they are oftenaccompanied by other polysaccharides such as chitosan. Chitosan is alinear polysaccharide composed of randomly distributed chains of β-(1-4)D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine(acetylated unit).

In some alginate formulations albumin can be used as the substance ofcharge, preferably sterile and pyrogen-free human albumin, which canalso act as a protector of the active ingredient in the process ofmanufacture or as a stabiliser during the long-term conservation of theproduct.

The active ingredient which release in plasma is intended to be modifiedcan be a complex and labile active ingredient. More specifically, theactive ingredient features exhibits biological activity. This biologicalactivity can be developed through enzymatic activity, transport,molecular interaction or binding with a ligand. In both cases, it wouldbe a question of active ingredients labile or sensitive to energeticconditions of manufacture in temperature, pressure and/or nonpolarenvironments amongst others, since small structural changes can lead toan irreversible loss of biological activity.

As examples of active ingredients with biological activity, humanpeptide hormones such as melatonin, serotonin, thyroxin, epinephrine,norepinephrine, dopamine, adrenocorticotropic hormone, angiotensinogenand angiotensin, vasopressin, atriopeptin, calcitonin, erythropoietin,follicle stimulating hormone, glucagon, human chorionic gonadotropin,human placental lactogen, growth hormone, inhibin, insulin, insulin-typegrowth factor (or somatomedin), luteinising hormone,melanocyte-stimulating hormone, oxytocin, prolactin, thrombopoietin,neuropeptide Y, histamine, together with their derivatives can bementioned.

Other examples can be biologically active proteins such as albumin,alpha 1-antitrypsin, alpha-acid glycoprotein, alpha-2-macroglobulin,antithrombin, haptoglobin, ceruloplasmin, lipoproteins, transferrin,plasminogen, fibrinogen, complementary proteins, clotting factors, andimmunoglobulins, amongst others.

The fact that these active ingredients are biologically active makesthem especially vulnerable to a possible loss of functionality as aresult of minor structural damage. This structural damage can beassociated with temperature, pressure, polarity of the medium,osmolality, presence of oxygen, agitation, etc.

In this connection, clotting factor VIII stands out amongst these activeingredients because of its extreme lability. Due to its structuralcomplexity, it is very difficult to adequately stabilise the biologicalactivity of FVIII, especially in its purified form. For example, Parti Ret al (Haemophilia 2000; 6: 513-522) explain how even in its lyophilisedform, the biological activity of FVIII begins to be compromised attemperatures of above 40° C. This instability is most evident when FVIIIis in solution, where even at 25° C. signs of instability are observed.In the case of Factor IX and of Factor VIIa sensitivity to externalfactors such as temperature is also known.

In this regard it must be noted that the manufacturing process appliedallows therapeutic preparations with biological activity of FVIII to beobtained. This means that the method is applicable to active ingredientsexhibiting biological activities which are difficult to stabilise, and,therefore, that the present invention is applicable to ingredients whichare as labile as FVIII. By extension, the present invention isapplicable to ingredients that are more stable than FVIII. As a result,clotting factors are a clear example of an active ingredient which canbenefit from the application of the formulation as described in thepresent invention.

In the present invention the active ingredient included in the polymermicrosphere can thus be a peptide, a protein or a hormone exhibitingbiological activity. Preferably, the active ingredient is a clottingfactor and more preferably, the active ingredient is the VIII factor,the von Willebrand factor, the complex formed by the VIII factor and thevon Willebrand factor, the IX factor or the VIIa factor.

These ingredients can be of human, animal, recombinant or transgenicorigin. In the latter cases, the synthesised molecule can be areproduction of the natural molecule or be deliberately modified.

Obtaining the Composition

Microencapsulation is a process of coating molecules, solid particles orliquid globules, with materials of a different nature, in order tocreate particles of micrometric size. The products resulting from thistechnological process are named microparticles, microcapsules ormicrospheres.

There are several microencapsulation techniques:

-   -   Microencapsulation by chemical methods:        -   Interfacial polymerisation    -   Microencapsulation by physicochemical methods:        -   Evaporation of solvent        -   Coacervation        -   Gellification        -   Chelation        -   Formation of vesicles    -   Microencapsulation by mechanical methods:        -   Extrusion        -   Co-extrusion        -   Spray drying        -   Spray chilling

The chosen technique for the manufacture of microparticles described inthe present invention is spray drying, as described in Erdinc B. I.[Erdinc B. I. (2007) Micro/nanoencapsulation of proteins withinalginate/chitosan matrix by spray drying, Degree Thesis, Queen'sUniversity, Kingston, Canada]. This manufacturing technique features asingle stage and microparticles are obtained as the final product.

The manufacturing process of a biocompatible composition for intravenousadministration which includes microparticles of alginic acid or itssalts for the controlled release of an active ingredient of the presentinvention is characterized by the stages of:

-   -   spraying, in which the solution/suspension/emulsion containing        the active ingredient and the polymer is pumped through a nozzle        and is dispersed in the form of drops,    -   drying in the drying chamber, where the hot air assists the        evaporation of the solvent from the drops, and    -   collection of the encapsulated product        this procedure being performed at a temperature of between 140        and 180° C. with a supply flow rate between 35 and 40 m³/h, an        injection flow rate between 3.5 and 5 ml/min and a pressure        between 4 and 6 psi.

Under these conditions it is possible to obtain particles with a size ofless than or equal to 5 μm, preferably between 1 and 4.5 μm and maintainthe activity of the active ingredient. In addition, the average size ofthe particles can be improved in an optional process of homogenisationof the emulsion before the spray stage. This additional homogenisationprocess is carried out by means of pressure, for example between 1500and 2000 psi.

The encapsulation of active ingredients by means of spray drying is acontinuous process in which a solution or emulsion is dehydrated,recovering a solid formed by microparticles at the end of the process.

To this end, the fluid containing the active ingredient is drivenmechanically at a predetermined injection flow rate towards a nozzle orrotating disk in which it is sprayed in millions of very small drops.The size of the drops is determined in large measure by the pressure ofthe gas that causes the spray of the fluid. This process takes place ina closed chamber where a stream of controlled gas, which is usually air,circulates continuously at a predetermined speed of intake and at acontrolled temperature.

As a result of the spraying, the fluid greatly increases its contactsurface area with the air, so that when faced with the current of dryingair there is a rapid evaporation of the fluid solvent, usually water.This rapid evaporation causes the internal cooling of each small dropdue to the heat needed for the change in state. In this way it ispossible to carry out fast drying whilst minimising the thermal shock tothe active ingredient. Upon completion of the process, the product iscollected in solid form.

Description of the Composition

The microparticles obtained are distinguished by determining theirparticle average size, their Z potential and biological activity. Thesize of particle is determined with a Beckman Coulter LS13320 device bya diffraction laser.

As it is a question of intravenous administration, it is necessary forthe particle size to be less than or equal to 5 μm, preferably between 1and 4.5 μm, because higher particle sizes could cause the formation ofthrombi.

The Z potential, which is determined with a Malvern Zetasizer device, isone of the fundamental parameters controlling the interaction of theparticles in suspension. It is determined by the nature of the particlesurface and the dispersion medium. In this case the optimal values arethose above −30 mV since this ensures repulsion between particles andabsence of aggregates. It has been shown that microparticles with Zpotentials close to 0, preferably between −30 mV and 0, have low liveruptake and cell clearance levels. (Szycher, Michael, High PerformanceBiomaterials: A Comprehensive Guide to Medical and PharmaceuticalApplications, published by CRC Press, 1991 ISB 0877627754,9780877627753, 812 pages).

Use of the Composition

The pharmaceutical forms of modified or controlled release are thosedesigned in such a way as to change the speed and/or the place ofrelease of the active substance or substances in relation to thepharmaceutical form of conventional release, administered in the sameway.

In the present invention it has been observed how the encapsulation ofactive ingredients exhibiting biological activity, such as proteins, andmore specifically, clotting factors, allows a controlled release in anin vitro release model. Factor VIII is notable for its extremesensitivity to external factors given its structural complexity. Infact, even freezing FVIII in human plasma itself, its natural matrix,causes a partial loss of biological activity (Bravo, M. I. et al,Pharmeuropa Scientific Notes, 2006-1 pp. 1-5).

So when the microparticles containing human FVIII described in thepresent invention are placed in a continuous flow cell in a similarenvironment to human plasma, a delay has been observed, compared withthe unencapsulated product, in the release of FVIII in the medium.

Similarly, intravenous administration of FVIII-containing microparticlesof the present invention in rabbits, results in consistent andsignificant extension of the half-life of FVIII in plasma, as comparedto the conventional product. Furthermore, no adverse effects wereobserved in animals that might indicate a toxic effect associated withthe formulation described.

The in vivo pharmacokinetics data are very significant because theyprove without doubt that the effect of opsonisation and accelerateduptake for the MPS has been dealt with properly for the formulation ofthe invention.

The present invention can be used in the treatment of variouspathologies that require the intravenous administration of complexingredients, which can include for example, bleeding disorders andclotting disturbances, hormonal disorders, etc. In these cases, asignificant extension of half-life would be achieved, which for examplein the case of FVIII, could include reducing the number ofadministrations for maintaining a primary prophylaxis regime, forexample, weekly administration.

A possible drawback associated with the use of hydrophilic polymers maybe the partial dissolution of the microparticle during the period oftime between suspension of the product in an aqueous vehicle ofadministration, for example, water for non-pyrogenic and sterileinjection and the time of the intravenous infusion. This type ofdisadvantage can be overcome for example with the use of partiallyapolar biocompatible solutions, such as ethanol, propylene glycol,polyethylene glycol, dimethylsulphoxide, N-methyl-2-pyrrolidone,glycofurol, isopropylidene-glycerol, glycerol formal or acetone (Mottu Fet al. Journal of Pharmaceutical Science & Technology 2000 Vol. 54, No.6, 456-469), amongst others, as vehicles of resuspension andadministration of the microparticles described in the present invention.

The invention can be produced, for example, in the form of a dehydratedor freeze-dried product packed in a vacuum or inert atmosphere, allowinglong-term stability in varying temperature conditions, for example,between 2° C. and 40° C. The product thus preserved can be administeredintravenously after reconstitution with a solvent which can be water forinjection, or a saline solution, or a mixture or an aqueous salinesolution with a variable content, for example between 0.5% and 50% ofbiocompatible solvents such as for example ethanol, propylene glycol,polyethylene glycol, dimethylsulphoxide, N-methyl-2-pyrrolidone,glycofurol, isopropylidene-glycerol, glycerol formal or acetone, amongstothers.

Advantages Over the Prior Art

The present invention describes the production of hydrophilicmicroparticles of alginate with a combination of a size suitable forintravenous infusion and physicochemical features suitable forpreventing their rapid phagocytosis, allowing an extension of thehalf-life of complex active ingredients.

Alginate is biocompatible and is eliminated via urine, and has noassociation with any known effect of toxicity. Due to its features, thepresent invention is compatible with the administration of proteins andcomplex active ingredients.

This invention can overcome all the disadvantages that have made acontrolled administration intravenous system impractical, thusdecreasing the number of administrations necessary for treatment withunchanged active ingredients for intravenous use. In this regard itshould be noted that the present invention does require any modificationof an active ingredient, in the amino acid sequence, glycosylations orintroduction of synthetic derivatives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparative graph of the results of the in vitro releasetests of BATCH 9 and BATCH 1.

FIG. 2 shows the pharmacokinetics of human FVIII:C in rabbit plasmaafter the administration of unencapsulated FVIII and after theapplication of the composition.

FIG. 3 shows the pharmacokinetics of human VWF:Ag in rabbit plasma afterthe administration of unencapsulated FVIII and after the application ofthe composition.

EXAMPLE 1 Preparation of the Microparticles

The spray drying process has been used for the production of alginatemicroparticles as described in Erdinc B. I. [Erdinc B. I. (2007)Micro/nanoencapsulation of proteins within alginate/chitosan matrix byspray drying, Degree Thesis, Queen's University, Kingston, Canada].Basically, microparticles were prepared by producing an emulsion withthe polymer and the active ingredient chosen.

A Büchi Mini Spray Dryer B-290 device was used for spraying the samplesunder the following conditions: spray temperature: 140° C.-180° C.,intake rate: 35-40 m³/h, injection flow rate: 3.5-5 ml/min and pressure4-6 psi.

EXAMPLE 2 Description of the Microparticles

Tables 1, 2 and 3 describe the materials used in the manufacture ofmicroparticles and their features, including size, Z potential andyield. The manufacturing process and the conditions used were asdescribed in Example 1.

TABLE 1 Description of FVIII microparticles (plasmatic FVIII) Meanparticle size Z Potential Batch Polymer (μm) (mV) BATCH 1 SodiumAlginate 3.6 −32 FVIII BATCH 2 Sodium Alginate 4.5 −32 FVIII BATCH 3Sodium Alginate 4.7 −31 FVIII

The FVIII activity/FVIII antigen ratio gives an idea of the proportionof active protein in a given sample. In this way, if we compare theactivity/antigen ratio in the initial sample with that obtained in theencapsulated sample, we can calculate the proportion of activeingredient which remains functional after microencapsulation. In theexample, we found that the activity yields during the process ofencapsulation, expressed as a percentage compared to the initialactivity yield, are 57.6%, 33.9% and 35.7% for batches 1, 2 and 3respectively.

TABLE 2 Description of FIX microparticles (plasmatic FIX) Mean particlesize Z Potential Batch Polymer (μm) (mV) BATCH 4 Sodium Alginate 4.9 −63FIX BATCH 5 Sodium Alginate 4.5 −18 FIX BATCH 6 Sodium Alginate 4.9 −10FIX

In this case, we found that the activity yields during the process ofencapsulation in batches 4, 5 and 6, are 100% in all said batches.

TABLE 3 Description of rFVIII microparticles (_(recombinant)FVIII) andrFVIIa (_(recombinant)FVIIa) Mean particle size Z Potential BatchPolymer (μm) (mV) BATCH 7 Sodium Alginate 4.7 −70 rFVIII BATCH 8 SodiumAlginate 4.9 −64 rFVIIa

In the case of proteins of recombinant origin, the activity yieldsdetermined during the process of encapsulation were of 25% and of 71%for batches 7 and 8 respectively.

In all batches, the size of particle was determined with the BeckmanCoulter LS13320 device through a diffraction laser and the Z Potentialwas measured with the Malvern Zetasizer device.

The biological activity of FVIII was determined by deficient plasmaclotting assay or by evaluating the generation of FXa by chromogenesis.In the case of FVIIa and FIX, the biological activity was determined byevaluating the clotting time (partial activated thromboplastin time) ofplasmas without FVII and FIX, respectively. The protein concentrationwas determined by the immunological detection method of enzyme-linkedimmunosorbent assay (ELISA) using specific antibodies against FVIII:Ag,FIX:Ag or FVII:Ag respectively.

The activity/antigen ratios, indicative of the proportion of activeprotein in a given sample were calculated by obtaining the quotientbetween the activity and antigen units for the specific activeingredient in the sample. The calculation of the activity/antigen yieldis carried out by estimating the percentage of variation between theactivity/antigen ratios of the starting sample and of the finalencapsulated product.

As can be seen in all cases, the average particle size is less than orequal to 5 μm and the Z Potential is negative. Also the results ofactivity/Ag yield indicate that the biological activity during theprocess is being maintained.

The various tables show that the controlled release system is suitablefor different active ingredients.

EXAMPLE 3 In Vitro Release Test

A controlled release test with a continuous flow cell is performed in aSotax CE1 device in closed circuit in order to evaluate the release ofactive ingredient.

The test was conducted at a temperature of 37° C. with a flow rate of7-25 ml/min using as a dissolving medium an imidazole pH 7.3 buffercontaining 1% human albumin. A representative sample was extracted foranalysis at different times (5 minutes, 10 minutes, 15 minutes, 30minutes, 60 minutes, 120 minutes, 180 minutes and 240 minutes). Thevolume of extracted sample was replaced with the same volume of freshmedium in order to correct the loss of volume.

The biological activity of FVIII was determined by a deficient plasmaclotting assay or by evaluating the generation of FXa by chromogenesis.In the case of FVIIa and FIX, the biological activity was determined byevaluating the clotting time (partial activated thromboplastin time) ofplasma without FVII and FIX, respectively. The protein concentration wasdetermined by the immunological detection method of enzyme-linkedimmunosorbent assay (ELISA) using specific antibodies against FVIII:Ag,FIX:Ag or FVII:Ag respectively.

After completion of the test the following results were obtained:

TABLE 4 In vitro release test of unencapsulated lyophilised FVIII (BATCH9) BATCH 9 (unencapsulated) Time (min) FVIII: C released (%) 5 100

TABLE 5 In vitro release test of FVIII nanoparticles (BATCH 1) BATCH 1(encapsulated) Time (min) FVIII: C released (%) 5 20.7 10 29.6 15 35.230 40.5 60 51.7 120 63.0 180 69.0 240 71.0

We can see that the composition of the microparticle applied to theactive ingredient modifies the release kinetics of the product comparedto unencapsulated product.

EXAMPLE 4 Pharmacokinetics of Factor VIII in Animals

In order to evaluate the effect of the composition on the release ofactive ingredient in vivo, a pharmacokinetics test was carried out onrabbits. For this, a dose of 50 IU/kg of human FVIII from Batch 9 (notencapsulated) was administered intravenously to three female New ZealandWhite rabbits. Similarly, a dose of 50 IU/kg of encapsulated FVIII fromBatch 1 as manufactured as described in example 1 and describedaccording to example 2 was administered intravenously to a further threefemale New Zealand White rabbits. At various times, plasma samples wereobtained which were analysed to detect the presence of human FVIII:C, asdescribed in Table 6. The detection of human FVIII was performed bychromogenesis after selective immunological capture of the human FVIIImolecules. This allows the activity of infused human FVIII to bedistinguished from that of rabbit FVIII.

TABLE 6 Pharmacokinetics of human FVIII: C in rabbit plasma after theadministration of unencapsulated FVIII and after the application of thecomposition FVIII FVIII microparticles (unencapsulated) (encapsulated)Time BATCH 9 BATCH 1 (hours) hFVIII: C (U/ml) hFVIII: C (U/ml) 0 0.018 ±0.024 0.046 ± 0.012 0.5 0.931 ± 0.069 0.459 ± 0.186 2 0.678 ± 0.2360.534 ± 0.158 6 0.238 ± 0.165 0.346 ± 0.076 12 0.054 ± 0.062 0.243 ±0.005 24 0.023 ± 0.027 0.090 + 0.008 36 0.022 ± 0.024 0.073 ± 0.009 490.021 ± 0.026 0.033 ± 0.011

We can see from the results that the composition delays the release ofthe active ingredient in plasma. In addition, these results demonstratethat there is no cell mechanism (liver, spleen, or macrophages) whichrapidly removes the microparticles from the circulation, in spite oftheir size.

The analysis of this data using appropriate software for this purpose(WinNonlin 5.2) allowed us to calculate the pharmacokinetic constantsdetailed in table 7.

TABLE 7 Pharmacokinetic parameter of human FVIII: C in rabbit plasmaafter the administration of unencapsulated FVIII and after theapplication of the composition FBI FVIII microparticles (unencapsulated)(encapsulated) BATCH 9 BATCH 1 FVIII: C Half-life (h) 3.0 ± 1.6 12.7 ±2.7 Average residence 5.1 ± 1.1 17.4 ± 3.8 time (h)

EXAMPLE 5 Pharmacokinetics of the Von Willebrand Factor in Animals

Both in the case of the BATCH 9 preparation (unencapsulated FVIII) andin the preparation of Batch 1 (encapsulated FVIII), the FVIII was ofplasma origin with a significant content of von Willebrand factor (VWF).This means that the encapsulation of the VWF occurs at the same time asthe encapsulation of FVIII. For this, their behaviour can be studiedindependently. For this we proceeded to independently analyse the VWFpharmacokinetics, by assessing the presence of the human VWF antigen(VWF:Ag) in the rabbit plasma. The results are shown in Table 8.

TABLE 8 Pharmacokinetics of human VWF: Ag in rabbit plasma after theadministration of the unencapsulated VWF and after the application ofthe composition FVIII/VWF FVIII/VWF microparticles (unencapsulated)(encapsulated) Time BATCH 9 BATCH 1 (hours) VWF: Ag (Ul/ml) VWF: Ag(Ul/ml) 0 0.000 ± 0.000 0.000 ± 0.000 0.5 0.859 ± 0.193 1.053 ± 0.048 20.552 ± 0.247 0.862 ± 0.055 6 0.150 ± 0.080 0.384 ± 0.106 12 0.033 ±0.022 0.207 ± 0.031 24 0.005 ± 0.002 0.040 ± 0.005 36 0.001 ± 0.0000.019 ± 0.008 49 0.001 ± 0.000 0.009 ± 0.005

We can see from the results that the composition delays the release ofthe active ingredient in plasma. In addition, these results demonstratethat there is no cell mechanism (liver, spleen, or macrophages) whichrapidly removes the microparticles from the circulation, in spite oftheir size.

The analysis of this data using appropriate software for this purpose(WinNonlin 5.2) allowed us to calculate the pharmacokinetic constantsdetailed in Table 9.

TABLE 9 Pharmacokinetic parameter of human VWF: Ag in rabbit plasmaafter the administration of unencapsulated FVIII/VWF and after theapplication of the composition Microparticles FVIII of FVIII(unencapsulated) (encapsulated) BATCH 9 BATCH 1 VWF: Ag Half-life (h)5.7 ± 0.3 11.1 ± 2.8 Average residence 3.6 ± 0.5 11.9 ± 3.7 time (h)

As can be observed, the encapsulation of the active ingredient, VWF inthis case, significantly extends its half-life.

While the invention has been described for examples of preferredembodiments, these should not be considered limitative of the inventionwhich will be defined by the broader interpretation of the followingclaims.

1. A biocompatible composition for intravenous administration comprisingmicroparticles of alginic acid or salt thereof less than or equal to 5μm in size and having a negative Z potential, and wherein the alginicacid or salt thereof is complexed with a therapeutic active ingredient.2. A composition according to claim 1, wherein the size of themicroparticles is between 1 and 4.5 μm.
 3. A composition according toclaim 1, wherein the Z potential is between −70 and 0, not including 0.4. A composition according to claim 1, wherein the active ingredient isa peptide, a protein or a hormone.
 5. A composition according to claim4, wherein the active ingredient is of human, animal, recombinant ortransgenic origin.
 6. A composition according to claim 4, wherein theactive ingredient exhibits labile biological activity.
 7. A compositionaccording to claim 4, wherein the active ingredient is a blood clottingfactor.
 8. A composition according to claim 4, wherein the activeingredient is factor VIII.
 9. A composition according to claim 4,wherein the active ingredient is VWF.
 10. A composition according toclaim 4, wherein the active ingredient is the complex formed by FVIIIand VWF.
 11. A composition according to claim 4, wherein the activeingredient is factor IX.
 12. A composition according to claim 4, whereinthe active ingredient is factor VIIa.